The pseudouridine synthases, found in all organisms, catalyze the isomerization of particular uridine residues in RNA to pseudouridine. Specific physiological roles for pseudouridine have also recently emerged: 1) pseudouridine is required in U2 snRNA for functional assembly of the U2 snRNP, which splices pre- mRNA; 2) the disease dyskeratosis congenita arises from the absence of a protein with sequence similarity to one pseudouridine synthase, TruB; 3) inhibition of pseudouridine synthases partly accounts for the cytotoxicity of 5-fluorouracil, an anti-cancer drug that is converted to 5-fluorouridine and incorporated into RNA in vivo. The mechanistic investigation of two pseudouridine synthases, TruB and RluA, is proposed. Events at the active site will be elucidated by integration of the information provided by site- directed mutagenesis, chemical modification, determination of kinetic isotope effects, and the direct observation of the adduct between pseudouridine synthases and RNA containing 5- fluorouridine using NMR methods. Site-directed mutagenesis revealed the catalytic importance of an aspartic acid, and site- specific chemical modification will probe the catalytic role played by that critical aspartic acid. Kinetic isotope effects will delineate the reaction step(s) affected by this amino acid. The chemical structure of the adduct between RNA containing 5- fluorouridine and the mutant protein will be determined and compared to that with the wild-type enzyme. This comparison will reveal the nature of protein-RNA interaction by identifying the particular groups in both the protein and the RNA that participate in the interaction, which is critical for understanding both pseudouridine synthase catalysis and the action of 5-fluorouracil. These studies will be expanded to examine more amino acid residues implicated in catalysis, including cysteine residues and the amino acids of TruB that align with the protein whose absence leads to dyskeratosis congenita. Taken together, the observations from these diverse techniques add up to more than the sum of their parts, combining to generate a fuller understanding of events at the active sites of pseudouridine synthases, enzymes whose physiological importance is just beginning to be fully appreciated.